Plasmodium parasites. Plasmodium relies on diverse microtubule cytoskeletal structures for its reproduction, multiplication, and dissemination. Due to the small size of this parasite, its cytoskeleton has been primarily observable by electron microscopy (EM). Here, we demonstrate that the nanoscale cytoskeleton organisation is within reach using ultrastructure expansion microscopy (U-ExM). In developing microgametocytes, U-ExM allows monitoring the dynamic assembly of axonemes and concomitant tubulin polyglutamylation in whole cells. In the invasive merozoite and ookinete forms, U-ExM unveils the diversity across Plasmodium stages and species of the subpellicular microtubule arrays that confer cell rigidity. In ookinetes, we additionally identify an apical tubulin ring (ATR) that colocalises with markers of the conoid in related apicomplexan parasites. This tubulin-containing structure was presumed to be lost in Plasmodium despite its crucial role in motility and invasion in other apicomplexans. Here, U-ExM reveals that a divergent and considerably reduced form of the conoid is actually conserved in Plasmodium species.Malaria is caused by unicellular  An expansion microscopy study of Plasmodium species, parasites that cause malaria, unveils novel cytoskeletal features including a remnant conoid - a tubulin structure that was presumed to be lost in these organisms despite its crucial role in host cell invasion by related apicomplexan parasites. Plasmodium parasites that belong to the phylum Apicomplexa. More than 6,000 species have been described in this phylum [Toxoplasma gondii and Cryptosporidia. During their life cycle, apicomplexan parasites undergo multiple differentiation steps into morphologically distinct forms sustaining either (i) sexual reproduction; (ii) asexual replication; or (iii) dissemination via egress and invasion of host cells [Malaria is caused by unicellular s phylum  includinst cells . Each ofPlasmodium zoites [T. gondii and gregarines, possess a conoid [Plasmodium has been traditionally considered to lack a conoid and belongs to the Aconoidasida [T. gondii [Plasmodium.A unique cytoskeletal feature that distinguishes Apicomplexa from other eukaryotes is the apical complex. This complex is assembled in polarised invasive stages, called zoites. It includes specialised secretory organelles and a microtubule organising centre (MTOC) called the apical polar ring (APR). The secretory organelles release various factors necessary for egress, motility, and invasion . An arram zoites ,5. The fm zoites  has defia conoid . Based ooidasida . While t. gondii , its molPlasmodium microtubule-based structures heavily relies on electron microscopy (EM). Fluorescent light microscopy is instrumental to complement EM with the possibility of using multiple markers to infer the dynamic and the molecular composition of Plasmodium cytoskeletons. However, owing to the small size of the parasites, subcellular imaging by fluorescent light microscopy still poses a major challenge in Plasmodium. Super-resolution techniques have recently been implemented, but structured illumination microscopy (SIM) provides only slightly improved resolution (approximately 120 nm) in comparison to diffraction-limited light microscopy (approximately 240 nm), while stimulated emission depletion (STED) microscopy can reach a resolution of up to 35 nm [Plasmodium microtubule cytoskeleton remain difficult to interrogate.Our understanding of to 35 nm ,11. Howeto 35 nm . The impto 35 nm . BecausePlasmodium cells in an isotropic fashion by ultrastructure expansion microscopy (U-ExM) [T. gondii [Plasmodium stages and species including 2 zoite stages, Plasmodium falciparum schizonts, Plasmodium berghei schizonts, and P. berghei ookinetes, as well as developing P. berghei microgametes. We show that U-ExM resolves the structure of the axonemes, the mitotic hemispindles as well as the subpellicular microtubules. Importantly, it reveals the existence of an apical tubulin ring (ATR) colocalising with markers of the conoid in other parasites that is atop the APR of motile ookinetes, suggesting that the remnant of a conoid was retained in this genus. In conclusion, U-ExM enables visualisation of cytoskeletal structures at a nanoscale resolution in Plasmodium and represents an accessible method without the need of specialised microscopes.Here, we reasoned that physical expansion of  (U-ExM)  could pr. gondii ,16. We ePlasmodium, we compared protocols for fluorescent microscopy of tubulin structures with or without expansion on multiple life cycle stages . Briefly, Plasmodium parasites were first sedimented on poly-D-lysine coated coverslips prior to cold methanol fixation. Next, proteins of the samples were anchored to a swellable polymer, followed by denaturation and expansion. Immunostaining was subsequently performed post-expansion as previously described [Fig 1A).To test the potential of U-ExM  to imageescribed  P. berghei microgametocytes into microgametes, a process that is essential for sexual reproduction and establishment of infection in the mosquito . During microgametogenesis, the component parts of 8 axonemes are assembled within the gametocyte cytoplasm to form flagellated male gametes in a process called exflagellation. Confocal imaging of cells stained for α/β-tubulin showed a bundle of undistinguishable axonemes coiled around the octoploid nucleus. In contrast, expanded gametocytes resolved individual axonemes , featuring the gain of resolution obtained using U-ExM. Importantly, comparison of the average diameters of cells with or without expansion indicated an approximately 4.2-fold size increase in the linear dimension, from around 5 to 20 μm .We first imaged the development of P. berghei ookinete, a large motile extracellular zoite of 10 to 12 μm in length, responsible for colonisation of the mosquito midgut that displays a subpellicular microtubule network radiating from the APR. Non-expanded ookinetes stained for α/β-tubulin showed a diffuse cytoplasmic signal with an enrichment at the cell periphery and no visible microtubule structures . Expanded cells showed an approximately 4.2-fold increase in both width and length, highlighting the isotropic expansion of the specimen . This resulted in the clear resolution of individual subpellicular microtubules that radiate from the APR .We then applied U-ExM to P. falciparum. During erythrocytic schizogony, the parasite undergoes multiple rounds of replications to form up to 32 merozoites per schizont. Merozoites are approximately 1 to 2 μm long zoites that upon rupture of the host erythrocyte quickly reinvade erythrocytes. Confocal imaging of late stage schizonts prior to expansion showed a punctuate signal for α/β-tubulin . Sample expansion showed a 4.3-fold increase in global size without apparent major morphological distortions and highlighted the previously described mitotic hemispindles [S1 Fig).Next, we stained schizonts of spindles   and demonstrates that U-ExM can be readily applied to multiple Plasmodium stages, revealing subtle details of its internal cytoskeleton organisation.Altogether, expansion of 3 stages from 2 Plasmodium assembles a flagellum exclusively prior to the formation of the microgamete in the midgut of the mosquito vector [Plasmodium is remarkably different from known models [o vector . Microgao vector ,19. The o vector . Flagelln models . Axonemen models , and then models . EM obsen models . As the n models .Fig 2A–2D). To do so, we immunolabelled for α/β tubulin microgametocytes that were fixed 15 minutes after gametogenesis activation, when the first exflagellation events are observed. This revealed snapshots of axonemal assembly that have only been visible by EM observations of serial sections. We observed a wide range of patterns, ranging from incomplete axoneme formation where fan-shaped arrays of short microtubules radiate from the basal body  to apparently fully assembled axonemes . A detailed analysis of 25 whole-cell projections revealed that only 4% (1/25) of 15-minute activated gametocytes showed fully formed axonemes, while the others cells presented 3 main and nonexclusive intermediate states. First, 80% of cells (20/25) showed less than 8 axonemes, which could reflect defects in basal body replication or other early impairment of microtubule polymerisation . Second, we observed that within single gametocytes, both incomplete and fully assembled axonemes coexist within a shared cytoplasm . Third, 44% (11/25) of the imaged axonemes displayed apparently free microtubule singlets or doublets . In contrast to the frequent observation of misassembled axonemes in developing gametocytes, the axoneme of free microgametes did not show such apparent defects . To ascertain that the observed structures correspond to non-assembled axonemes, we imaged Kin8B-KO microgametocytes where full-length singlet or doublet microtubules are formed but not assembled into the 9+2 pattern [Therefore, we undertook the analysis of axonemal assembly in cellulo in microgametocytes using U-ExM , although we also observed that some microtubules that were not incorporated into mature flagella were lacking PolyE . These results suggest that PolyE is added onto assembling axonemes and is more strongly detected in fully formed axonemes . To test whether PolyE is dependent on axonemal formation, we specifically stained Kin8B-KO microgametocytes and detected a signal of PolyE on microtubules from non-assembled axonemes as well as a high background in the cytoplasm of the gametocytes, possibly reflecting unincorporated polyglutamylated tubulin dimers or cross-reactivity with other glutamylated proteins whose distribution is altered in the mutant. Consistent with this, polyglutamylated tubulin was not affected in the mutant line . Altogether, these results suggest that PolyE deposition occurs in a timely manner during axoneme formation, but that is independent from complete assembly .To take advantage of the versatility of U-ExM, we then stained gametocytes for polyglutamylated tubulin a posttranslational modification that stabilises microtubules with various roles in regulating flagellar motility . PolygluPlasmodium gametogenesis that have been overlooked by conventional fluorescence microscopy.Altogether, U-ExM unveils details of axoneme formation in whole cells to levels that have only been accessible by EM. Based on these results, we anticipate that expansion microscopy will reveal new aspects of P. falciparum schizonts stained for α/β tubulin . Two distinct microtubule structures were observed in this stage by U-ExM: the mitotic hemispindles and the subpellicular microtubules. In late schizonts, we observed the hemispindles that form an array of microtubules, which radiate from single MTOCs, as revealed by centrin staining . Expansion of P. berghei segmenter schizonts revealed a similar pattern where PolyE is observed towards the apical end of the parasite. However, intracellular or free P. berghei merozoites contained up to 9 subpellicular microtubules . NHS-ester staining delineated the merozoite shape as well as contrasted structures with different protein density that likely correspond to the APR and the 2 secretory vesicles named rhoptries. As expected, the microtubules appeared to be located underneath the pellicle and nucleating from the APR, further confirming that they correspond to subpellicular microtubules. These differences between P. falciparum and P. berghei schizonts highlight the diversity of the apical complex organisation across species within the Plasmodium genus.In P. falciparum merozoites was shown to contain 3 electron dense concentric rings . A significantly larger number of subpellicular microtubules (>40) that radiate from the APR and covering most of the ookinete body were distinctly observed in expanded ookinetes, a number consistent with previous EM observations ranging from 47 to 59 [Fig 4A, S5 Fig). A less intense signal for PolyE was observed at the apical extremity of the ookinete, likely corresponding to the electron dense collar structure region [Fig 4A, S5 Fig).The apical complex of ar rings ), with tar rings ,5. The tasmodium . We thus47 to 59 –33. As ie region  . We then separately stained for α and β tubulin and found that both are present at the ATR, suggesting that it is composed by α-β heterodimers . The observation of the ATR was unexpected as such a ring of tubulin is reminiscent of a conoid found in coccidian but thought to be lost in the Haemosporidia order containing Plasmodium [T. gondii tachyzoites, the conoid is a central component of the apical complex and consists in a set of tubulin fibres that form a cone-shaped structure located between the APR and 2 additional apical rings called the preconoidal rings [Plasmodium.Importantly, the improved resolution of U-ExM in ookinetes allowed us to observe a ring of tubulin above the APR that we named the ATR . Of relevance, some proteins associated with the conoid in T. gondii [Plasmodium [Plasmodium ookinetes [Fig 4B). While the distance between the SAS6L-GFP/ATR ring and the APR was the same, the diameter of the SAS6L-GFP ring was slightly shorter suggesting that SAS6L-GFP or at least its carboxyl terminus is facing the interior of the ATR . However, this ring was larger and slightly above the ATR/SAS6L-GFP ring, further suggesting the presence of multiple apical rings in ookinetes . However, despite the presence of the ATR, no intra-conoidal microtubule could be observed.First, we noticed that the ATR is not polyglutamylated consistent with previous observations for the conoid of . gondii  . gondii , 35 are asmodium . This inokinetes . In expaokinetes  colocaliookinete  and was . gondii . U-ExM r. gondii  formed aT. gondii conoid lies in its ability to protrude through the APR upon stimulation of egress and motility [Fig 4F–4J). Ookinetes rely on the cGMP-dependent protein kinase G (PKG) to sustain productive secretion and gliding motility [− ookinetes, the ATR was in close proximity to the APR . The remaining mutant parasites showed a significantly reduced distance of 145 nm between the ATR and the APR . These results suggest that the position of the ATR relative to the APR depends on the activation of motility in ookinetes.Another characteristic of the motility . We thusmotility ,40. To dmotility ,41,42. IFig 5A, S8 Fig). Further staining for α/β tubulin revealed that the APR is located within the third upper end of the collar, while the ATR is likely in line with the IMC and the collar as inferred by EM . An additional protrusion was also observed above the ATR by NHS-ester staining, which may correspond to a small protrusion of the plasma membrane frequently observed by EM .To determine the position of both the APR and the ATR in a morphological spatial context, we labelled proteins of expanded ookinetes in bulk with NHS ester dye conjugates. NHS-ester staining delineated the ookinete shape as well as contrasted regions with different protein density that likely correspond to the collar, the apical termini of the inner membrane complex  and the subpellicular microtubules . For example, we show that posttranslational modifications such as PolyE occur in forming axonemes. We also observed different and nonexclusive states with either less than 8 axonemes per microgametocyte, axonemes with different lengths within a cell, or frequent misincorporation of microtubules towards the distal extremity of the axoneme. It remains unknown whether these observations reflect transient intermediate stages of assembly or terminal errors, as previously suggested [S2 Fig). Further experimental characterisations will be required to test these hypotheses.Our results demonstrate that U-ExM can be applied successfully to several uggested . HoweverFig 6B). First, the ATR colocalises closely with the SAS6L ring [T. gondii [T. gondii tachyzoites [T. gondii. This additional ring is composed of MyoB, which was proposed to fulfil, at least in part, a similar role as the conoid-associated myosin H in T. gondii [Plasmodium apical complex shows stage-specific variations in its composition, as previously proposed [P. berghei and P. falciparum merozoites.The most striking feature revealed by U-ExM was the presence of an ATR in ookinetes that most likely corresponds to a remnant conoid. The structure of the ATR appears significantly reduced or compacted compared with the spiralling tubulin fibres that form the conoid in other members of Apicomplexa. It remains unknown whether the ATR is of the same nature as the tubulin fibres forming the conoid that adopt a curvature that is not compatible with microtubules . AltogetS6L ring  that is . gondii ,35. Secohyzoites . Third, . gondii . Interes. gondii , the ATR. gondii , furtherproposed ,36. In lPlasmodium ookinete. As in T. gondii tachyzoites, no obvious structure between the APR and the ATR can be highlighted by NHS-ester staining or EM, and it remains unknown how the relative position between the ATR and the APR is maintained. We also observed that a small protrusion of the plasma membrane frequently observed by EM is above the ATR. It is possible that additional preconoidal ring proteins create this extension but that the associated structures are not as electron dense as known preconoidal rings in T. gondii tachyzoites or P. falciparum merozoites to be assigned as such by EM or NHS-ester U-ExM. In addition to SAS6L and MyoB, several other proteins associated with the conoid in T. gondii have been recently shown to be expressed in Plasmodium schizonts, ookinetes, and sporozoites [Plasmodium stages.While the ATR likely corresponds to a remnant conoid, its position in line with the IMC and the collar in ookinete is remarkable. This suggests that when the ATR moves away from the APR, the whole apex of the ookinete does so, including the IMC and the collar. It thus seems that the apical complex is internalised within the rozoites ,29 and mPlasmodium parasites retained a conoid that is significantly divergent and reduced compared to the best described T. gondii conoid, which likely prevented its identification. Our results also suggest that the tubulin structure that initially defined the conoid is not expressed in all Plasmodium zoites, raising new questions on the molecular nature of the conoid. It now appears that the conoid is likely a conserved organelle in Plasmodium. However, its molecular composition is more diverse than initially expected, and the physiological requirements associated with this diversity remain mysterious. It was suggested that the conoid plays a mechanical role in the invasion by enteric apicomplexan parasites that must penetrate the thick barrier of the intestinal epithelium of vertebrates [P. berghei and P. falciparum schizonts thus likely reflect adaptations of the apical complex to the different host cells or environment encountered by these zoites.We thus propose that tebrates . The obsPlasmodium apical complex that have not been characterised before. Moreover, expansion microscopy methods have been successfully coupled with super-resolution approaches, leading to a further increase in resolution [Plasmodium cytoskeleton and its molecular organisation. Finally, the recently developed membrane-based expansion microscopy protocols [Plasmodium. In summary, the use of expansion microscopy can nicely complement electron and super-resolution microscopy as it provides molecular details to the available structural information.Here, expansion microscopy reveals new features of the solution . Such aprotocols , 44 coulS1 Table.All primary and secondary antibodies used in this study can be found in All animal experiments were conducted with the authorisation numbers GE/82/15 and GE/41/17, according to the guidelines and regulations issued by the Swiss Federal Veterinary Office.P. berghei ANKA strain-derived [Mycoplasma pulmonis) and subjected to regular pathogen monitoring by sentinel screening. They were housed in individually ventilated cages furnished with a cardboard mouse house and Nestlet, maintained at 21 ± 2°C under a 12-hour light/dark cycle, and given commercially prepared autoclaved dry rodent diet and water ad libitum. The parasitaemia of infected animals was determined by microscopy of methanol-fixed and Giemsa-stained thin blood smears.-derived  clone 2.-derived  together-derived , SAS6L-G-derived , and Myo-derived  were gro-derived . Moreove3, 20% foetal bovine serum and 100 U/ml penicillin, 100 μg/ml streptomycin) on a Histodenz cushion made from 55% of the Histodenz stock and 45% PBS. Parasites were harvested from the interface and collected by centrifugation at 500 g for 3 minutes. Gametocyte production and purification was performed as previously described [Schizonts were purified from overnight in vitro culture , supplemented with 1 × protease inhibitor cocktail  on ice for 30 minutes. Proteins were detected using the primary antibodies α-tubulin  and α-PolyE  and secondary antibodies α-mouse  and α-rabbit . Ookinete cultures were performed as previously described [P. falciparum strain 3D7, a clone from the NF54 isolate [ isolate , was groOokinetes and schizonts were centrifuged at 1,000 rpm during 5 minutes at 24°C and resuspended in 500 μL of PBS 1X. Parasites were sedimented on poly-D-lysine  coverslips (150 μL/coverslip) during 10 minutes at room temperature (RT). To stop the activation of the gametocytes, the same protocol was used but keeping the parasites at 4°C. Then parasites were fixed in −20°C methanol during 7 minutes and prepared for U-ExM as previously published . BrieflyDirectly after antibody staining, the gel where incubated in NHS Ester  and diluted at 10 μg/mL in PBS for 1 hour and 30 minutes at RT on a rocking platform. The gels where then washed 3 times for 15 minutes with PBS-Tween 0.1% then expanded overnight in ultrapure water.Imaging was performed on a Leica Thunder inverted microscope (Germany) using 63X 1.4NA oil objective with Small Volume Computational Clearing mode to obtain deconvolved images. Three-dimensional stacks were acquired with 0.21 μm z-interval and x,y pixel size of 105 nm. Images were analysed and merged using ImageJ software.Confocal microscopy was performed on a Leica TCS SP8 (Germany) with a 63×/1.4-NA  oil immersion objective, using the HyVolution mode20 to generate deconvolved images, with the following parameters: “HyVolution Grade” at max resolution, Huygens Essential as “Approach,” water as “Mounting Medium,” and Best Resolution as “Strategy.”The diameter and distance between the ring of tubulin and the microtubules were always measured from dual staining experiments. After applying a maximum projection of the stack, we used the homemade plug-in pickCentrioleDim on Fiji software developed by M. Le Guennec to measure the peak-to-peak distance for each fluorescent channel for each parasite. The expansion factor of each gel was after applied to the value to obtain the real distance.To measure the length of microtubules, we used Fiji to draw a line to define our region of interest (ROI) following tubulin staining and determined its associated length. The expansion factor of each gel was after applied to the value to obtain the real size.P. berghei extracellular ookinetes were fixed with 2.5% glutaraldehyde  and 2.0% paraformaldehyde (Electron Microscopy Sciences) in 10 mM PBS pH 7.4 for 1 hour at room temperature. Pelleted cells were embedded in 3% low-melted agarose  in 10 mM PBS and dissected in small pieces in order of easier handling and to prevent loss of cells during subsequent processing steps. After extensive washing (5 × 5 minutes) in 0.1 M sodium cacodylate buffer pH7.4 (Sigma), samples were postfixed with 1% osmium tetroxide (Electron Microscopy Sciences) reduced with 1.5% ferrocyanide (Sigma) in 0.1 M sodium cacodylate buffer pH 7.4 for 1 hour at room temperature, followed by postfixation with 1% osmium tetroxide alone (Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer pH 7.4 for 1 hour at room temperature. After washing with double distilled water (2 × 5 minutes) samples were en-block post-stained with 1% aqueous uranyl acetate (Electron Microscopy Sciences) for 1 hour at room temperature. Samples were then washed with double distilled water for 5 minutes and dehydrated in graded ethanol series . Samples were then infiltrated at room temperature with Durcupan resin (Sigma) mixed 100% ethanol at 1:2, 1:1, and 2:1 for 30 minutes each step, followed by fresh pure Durcupan resin for 2 × 30 minutes and transferred into fresh pure Durcupan resin for 2 hours. Finally, samples were embedded in fresh Durcupan resin filled small thin-wall PCR tubes and polymerised and cured at 60°C for 24 hours. Ultrathin sections (60 nm) were cut with Leica Ultracut UCT microtome  and diamond knife (DiATOME) and collected onto 2 mm single slot copper grids (Electron Microscopy Sciences) coated with 1% Pioloform plastic support film. Sections were then examined and transmission electron microscopy (TEM) images collected using Tecnai 20 TEM (FEI) electron microscope operating at 80 kV and equipped with a side-mounted MegaView III CCD camera  controlled by iTEM acquisition software (Olympus Soft Imaging Systems).EM was performed as previously described . RBCs inS1 Data(XLSX)Click here for additional data file.S2 Data(TIF)Click here for additional data file.S1 FigA), and expanded (B); ookinetes, non-expanded (C), and expanded (D); schizonts, non-expanded (E), and expanded (F). Scale bar: 5 μm. (G) Ratio between the size before and after expansion and the average expansion factor (gel size/coverslip size). Averages and standard deviations are given in the table from at least 3 independent experiments for each stage. Related to Fig 1 . (H) Plot profile of the ROI extracted from 1 ookinete image stained for α/β-tubulin  showing the resolution limit of U-ExM. The example of ROI used for this plot profile is indicated in green on panel D. The average distance between 2 microtubules is of 137 nm +/− 17. The corresponding raw data can be found in Measurement skills to evaluate parasite size before and after expansion. Dotted lines represent the ROI used for the measurements for each parasite stage: microgametocytes, non-expanded ((TIFF)Click here for additional data file.S2 FigExpanded WT microgametocytes and gametes stained for α- and β-tubulin  and PolyE . Arrows show apparent differences between tubulin and PolyE staining. The white asterisk denotes fully formed axonemes. The white arrowhead indicates an exflagellated gamete. Scale bar: 5 μm. U-ExM, ultrastructure expansion microscopy; WT, wild-type.(TIFF)Click here for additional data file.S3 FigA) Wide-field representative image of an expanded microgametocyte stained for α- and β-tubulin  and PolyE . White arrows  indicate the position and orientation of the plot profiles shown in B to D. (B–D) Plot profiles across axonemes and tubulin structures/MTs showing a higher level of PolyE signal in complete axonemes in comparison with tubulin structures/microtubules (MT). (E) Western blot analysis showing tubulin and polyglutamylated tubulin in WT and P. berghei Kin8B-KO gametocytes 15 minutes post-activation by XA. The histograms show the relative values from 3 independent biological replicates. The raw data for B, C, D, and E can be found in ((TIFF)Click here for additional data file.S4 FigA–C) Merge wide-field image (A) of an expanded schizont stained for NHS-ester (grey) (B) and α- and β-tubulin  (C). Arrowheads indicate the position of the APR seen as a grey ring. Dotted square boxes corresponds to the insets. Scale bar: 4 μm. (D) Confocal image of a free expanded merozoite stained with NHS-ester (grey) and α- and β-tubulin . The arrowhead indicates the position of the APR seen as a grey ring. Scale bar: 2 μm. (E) Plot profile along the dotted line in D. Note that the measured APR have an average diameter of 183 nm +/ 12 (n = 13). The corresponding raw data can be found in ((TIFF)Click here for additional data file.S5 FigA) Representative confocal image of WT ookinete expanded using the U-ExM protocol and stained for α/β-tubulin  and PolyE . The 2 channels are represented independently. Scale bar: 5 μm. (B) Representative gallery of confocal images of WT ookinetes. Ookinetes were expanded using the U-ExM protocol and stained for α/β-tubulin  and PolyE . Scale bar: 5 μm. Insets show the delineated white-boxed region. The ATR is not polyglutamylated, while the subpellicular microtubules are PolyE positive. ATR, apical tubulin ring; U-ExM, ultrastructure expansion microscopy; WT, wild-type.((TIFF)Click here for additional data file.S6 FigA, B) Representative wide-field images of WT ookinete expanded using the U-ExM protocol and stained for α- (A) or β- (B) tubulin . Scale bar: 2 μm. ATR, apical tubulin ring; U-ExM, ultrastructure expansion microscopy; WT, wild-type.((TIFF)Click here for additional data file.S7 Fig− ookinete mutant. Ookinetes were expanded using the U-ExM protocol and stained for α/β-tubulin  and PolyE . Scale bar: 5 μm. The ATR is closely associated with the APR in contrast with WT ookinetes. APR, apical polar ring; ATR, apical tubulin ring; U-ExM, ultrastructure expansion microscopy; WT, wild-type.Representative confocal images of a GCβ(TIFF)Click here for additional data file.S8 FigA–C) Representative confocal image of an expanded ookinete stained for α/β-tubulin  and NHS-ester (grey). Merge (A), α/β-tubulin staining (B) and NHS-staining (C). Scale bar: 5 μm. NHS-ester staining reveals the position of the collar and the nucleus. Microtubules are only weakly detected, and micronemes are not stained by NHS-ester in contrast with the collar. NHS, N-hydroxysuccinimide.((TIFF)Click here for additional data file.S1 VideoThree-dimensional rendering of an expanded ookinete stained for α/β-tubulin  and NHS-ester (grey). NHS, N-hydroxysuccinimide.(MOV)Click here for additional data file.S1 TableAll cell lines as well as primary and secondary antibodies used in this study are listed, including the dilution used, the source or reference, and the catalogue number.(DOCX)Click here for additional data file. 8 Oct 2020Attachment108700_1_rebuttal_1882459_qhjlvn.pdfSubmitted filename: Click here for additional data file. 30 Oct 2020Dear Dr. Hamel, Thank you for submitting your manuscript entitled "Expansion Microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we will accept it without further review, but we will need to do some reporting and formatting checks first.For this, we need you to complete your submission by providing the metadata that is required. 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Mechanically, it was confirmed that lncRNA NR-104098 may effectively inhibit EZH2 transcription by directly binding to E2F1 and recruiting E2F1 to the EZH2 promoter. In addition, ATPR can significantly increase the expression of lncRNA NR-104098, whereas knocking down NR104098 can inhibit the inhibitory effect of ATPR on the proliferation and induction differentiation of AML cells. Taken together, these results lead to deeper insight into the mechanism of ATPR-induced AML differentiation and prevent proliferation by inhibiting EZH2 on the transcriptional level.Abundant evidence has illustrated that long non-coding RNA (lncRNA) plays a vital role in the regulation of tumor development and progression. Most lncRNAs have been proven to have biological and clinical significance in acute myeloid leukemia (AML), but further investigation remains necessary. In this study, we investigated lncRNA NR-104098 in AML and its specific mechanism. The microarray analysis was performed on NB4 cells. Based on the related analysis results, we identified that lncRNA NR-104098 is a suppressor gene that is significantly upregulated in AML cells. LncRNA NR-104098 could inhibit proliferation and induce differentiation in AML cells  As a very malignant hematological malignancy, acute myeloid leukemia (AML) accounts for a large proportion of leukemia. AML occurs due to the malignant proliferation of hematopoietic stem and progenitor cells . The patLncRNA is a non-coding RNA longer than 200 bp that does not have the ability to encode proteins . LncRNA To solve this problem, we used microarray analysis to investigate the expression of lncRNA in NB4 cells before and after ATPR induction. After ATPR-induced NB4 cells, 8662 lncRNAs and 9093 mRNAs with significant differential expression were screened. To explore the potential biological functions of these differentially expressed lncRNAs, we used Gene Ontology (GO) enrichment and Genes and Genomes (KEGG) for analysis. We established an lncRNA/mRNA co-expression network based on bioinformatic predictions and microarray results, and we then examined key transcription factors (TFs) associated with differentially expressed lncRNA. In addition, our results demonstrated the importance of a new lncRNA NR-104098 in the regulation of AML proliferation and differentiation.In this study, we demonstrated that the lncRNA NR-104098 expression level was downregulated in AML cells. LncRNA NR-104098 inhibited the expression of EZH2 by interacting with the TF E2F1 to achieve proliferation inhibition and differentiation induction of AML. Moreover, lncRNA NR-104098 acted as a key factor in ATPR-inhibited AML proliferation and induced differentiation.–2 mol/L) and kept at −20°C.ATPR (purity: 99.66%) was synthesized by the Pharmacology Laboratory of Anhui Medical University and dissolved in absolute ethanol  , penicillin (100 U/ml), and streptomycin (100 g/ml) −1640 medium.We purchased lncRNA NR-104098 shRNA and negative scrambled shRNA from Hanbio . First, NB4 and THP-1 cells were seeded in 24-well plates before being transfected. Then, 30 μl shRNA was added to each well, allowed to stand at room temperature for 15 min, and placed in a cell culture incubator; the medium was changed after 24 h. pEGFP-C3-NR104098 were also purchased from Hanbio .RNA samples were extracted from cells using chloroform and a Trizol reagent . SuperScript II  was used for the synthesis of Complementary DNA (cDNA). Amplification reactions were performed using a reaction system containing SYBR Green PCR Master Mix (Vazyme), 1 μL of cDNA, and amplification primers. β-Actin served as an internal control.Several primers were used:lncRNA NR-104098 forward, 5′-CCTGTATTTCTGCACCCG -3′;lncRNA NR-104098 reverse, 5′-GCATGTTCTCACTCACGC-3′;EZH2 forward, 5′-CCCTGACCTCTGTCTTACTTGTGGA-3′;EZH2 reverse, 5′-ACGTCAGATGGTGCCAGCAATA-3′;E2F1 forward, 5′-CGGCGATGTTACGACATTA-3′;E2F1 reverse, 5′-CTTGTGGTAGTCTAGTTCTTGC-3′;β-actin forward, 5′-CGCCGCCAGCTCACCATG-3′;β-actin reverse, 5′-CACGATGGAGGGGAAGACGG-3′;Cell Counting Kit 8 (CCK8)  was used to measure cell proliferation capacity. The treated cells were seeded at 5000 cells per well in a 96-well plate, followed by overnight culture. Subsequently, the absorbance at 450 nm per well was detected using a microplate reader (Thermo Fisher Scientific). The number of viable cells was tested every 24 h for 3 days.The treated cells were collected, and the culture medium was removed by centrifugation and then fixed with pre-chilled 70% ethanol overnight. RNaseA was added to each tube and incubated in a 37°C water bath for 30 min; 400 ul propidium iodide (PI) was then added. CytoFLEX  was used to detect the cell cycle, and cycle distribution analysis was then performed with ModFIT software.The treated cells were collected, the culture medium was removed by centrifugation, the cells were washed twice with PBS, and the cells were then incubated with CD11b (CD11b-PE/CY5) or CD14 (FITC) in the dark for 30 min on a shaker. CD11b and CD14 expression levels were measured using CytoFLEX .The experimental method has been described previously . BrieflyWe commissioned Hanbio to prepare the human EZH2 luciferase reporter plasmid. We tested it according to the dual luciferase reporter gene specification . The cells were transfected, and then the Firefly and Renilla luciferase activities were measured. The activity of Firefly luciferase was normalized by Renilla luciferase.Cells were harvested, and nuclear proteins were extracted and then resuspended in RIP buffer. Immediately afterward, the resuspended RIP buffer was divided into three groups (for input as well as co-precipitation of IgG and E2F1 antibodies). Then, the supernatant was collected by centrifugation, and IgG (Abcam) or human anti-E2F1 antibody (Abcam) was added to each and then incubated at 4°C for 2 h. Subsequently, protein A beads were added and incubated for 1 h at 4°C. After centrifugation, the cells were washed three times with RIP buffer, then once with PBS, and the beads were resuspended in Trizol. Finally, quantitative detection was performed by reverse transcription quantitative polymerase chain reaction (RT-qPCR).Biotin-labeled RNA pull-down was performed as described previously . Brieflyin situ hybridization (FISH) analysis, and cultured NB4 and THP-1 cells in NG medium containing lncRNA NR-104098  fluorescent probe as described previously  and then used this for the RT-qPCR assay.6) that were stably transfected with empty vector or pEGFP-C3-NR-104098 were injected subcutaneously. Eight weeks later, the mice were sacrificed, and the tumors were removed for weighing.Six-week-old male NCG nude mice were purchased from the Nanjing Model Animal Institute. The mice were allowed to adapt in the SPF environment of Anhui Medical University  for 1 week. The mice were randomly divided into two groups, and NB4 cells  followed by Duncan’s test. Differences were considered significant if the Microarray analysis was performed by OE Biotech , and the specific method was described before . We perfp-value < 0.05 (t-test). According to the results of We used microarray analysis to investigate whether induction of ATPR could affect lncRNA expression in NB4 cells. The intensity distribution was first normalized relative to the control, and volcano plots were then used to analyze differentially expressed lncRNAs and mRNAs . The resp < 0.05) (p < 0.05)  terms, 69 CC terms, and 62 MF terms that were upregulated ( < 0.05) , and 262 < 0.05) . Next, t < 0.05) . The mos < 0.05) ; the mos < 0.05) ; and the < 0.05) . In cont < 0.05) ; the mos < 0.05) , and the < 0.05) .p-value < 0.05, and these pathways were ranked based on the number of genes. Analysis results showed that 40 and 43 pathways are associated with upregulated and downregulated genes, respectively . Our CHIP analysis results show that overexpression of lncRNA NR-104098 can significantly improve the binding capacity of E2F1 . Our prein vivo, nude mice received subcutaneous injections of pEGFP-C3-NR104098 treated or empty vector NB4 cells to establish the tumor model. After 8 weeks, tumors were completely stripped. Photographs and weight of the tumors indicated that lncRNA NR-104098 overexpression cells grew much more slowly than empty vector group cells  RNA binding, and Ribosome. The lncRNA-mRNA co-expression network results showed that essential signal molecules  may be related to a variety of lncRNA, including NONHSAT131854, NR033856, LNST00000432120, and ENST00000495327. This suggests that lncRNA may play a critical role in AML proliferation and differentiation by regulating the expression of specific target mRNAs. MSH6 mutation is associated with the development of medulloblastoma or AML . BCAT1 aLeukemia is caused by a block in differentiation. The treatment of this disease is mainly induced differentiation therapy , b. Diffin vivo and in vitro. We also found that lncRNA NR-104098 inhibited EZH2 transcription by binding to the TF E2F1 and recruiting it to the EZH2 promoter, and silencing lncRNA NR-104098 can reverse the induction of AML by ATPR. These findings shed new light on a novel tumor-suppressing mechanism for lncRNA NR-104098 in AML. Although a large part of the function of lncRNA has yet to be developed, new research results showed that lncRNA can bind to TFs to activate or inhibit gene expression (Among all the significantly upregulated lncRNAs, we chose the most upregulated lncRNA NR-104098 for further study. We found that lncRNA NR-104098 expression levels were significantly reduced in AML cell lines compared to normal cell line expression levels. Mechanically, the effects of lncRNA NR-104098 overexpression and silencing on differentiation and appreciation were identified in order to study their relevance and the possible underlying mechanisms of their action. Furthermore, our results indicated that upregulation of lncRNA NR-104098 inhibits the proliferation and induces the differentiation of AML cells both pression . Previoupression . Resultspression . E2F actpression . Recent pression . Based opression . Comparepression . Our respression . In addipression . ATPR inpression . ATPR prpression . In thisOverall, for the first time, this study found that lncRNA NR-104098 may be an important regulator of AML. The results of subsequent mechanistic studies showed lncRNA NR-104098 on the transcriptional level of EZH2 by enhancing the binding of E2F1 to the EZH2 promoter . The intThe microarray data has been deposited to the GEO – GSE145168. Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.This study was approved by the Ethics and Research Committees of Anhui Medical University and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.in vivo research part. XP and FC optimized the manuscripts. All authors participated in this study, and read and approved the final manuscript.YF and LL conducted the project design. XX and MZ carried out the experiment. JL and SH participated in the bioinformatics analysis. TD, SZ, and YD performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-glycosylation. This subgroup presents various molecular abnormalities, of either the CDG-I or the CDG-II type, attributable to a lack of glycans or abnormal glycoform profiles, respectively. The most effective approach to identifying these N-glycosylation disorders is mass spectrometry (MS) using either released glycans, intact glycoproteins or proteolytic peptides as analytes. Among these, MS of tryptic peptides derived from transferrin can be used to reliably identify signature peptides that are characteristic of CDG-I and II. In the present study, matrix-assisted laser desorption/ionization (MALDI) MS was applied to various N-glycosylation disorders including ALG1-CDG, B4GALT1-CDG, SLC35A2-CDG, ATP6V0A2-CDG, TRAPPC11-CDG and MAN1B1-CDG. This method does not require the prior enrichment of glycopeptides or chromatographic separation, and thus serves as a practical alternative to liquid chromatography-electrospray ionization MS. The signature peptides are biomarkers of CDG.Congenital disorders of glycosylation (CDG), an increasingly recognized group of diseases that affect glycosylation, comprise the largest known subgroup of approximately 100 responsible genes related to  Among the various forms of CDG, those affecting N-glycosylation constitute a major subgroup, which is characterized by two types of molecular defects, i.e., a lack of glycans (CDG-I) or an abnormal glycan profile (CDG-II) (en bloc transfer of glycans to nascent proteins in the endoplasmic reticulum (ER). The defects of CDG-II occur in the subsequent trimming and decoration steps to form mature glycoforms in the ER and Golgi apparatus. In both types, patients also have unaffected glycoproteins, because the complete loss of mature glycans is incompatible with life.Congenital disorders of glycosylation (CDG) are an expanding group of disorders caused by defects in the glycosylation of proteins, and include more than 125 known genetic diseases.(CDG-II) . CDG-I iN-glycosylation of transferrin, and this observation contributed to establishing CDG as an entity. In the last thirty years, nearly 100 different disease types or genes responsible for congenital disorders of N-glycosylation have been identified. Since the symptoms and signs of CDG are quite diverse, making it impossible to establish a diagnosis using routine laboratory tests, MS is an essential approach for accurately diagnosing CDG.In 1992, mass spectrometry (MS) revealed a molecular abnormality derived from a defect in the early step of the  and each approach has its own advantages and disadvantages. An analysis of released glycans is well-established and applicable not only to transferrin but also to whole serum proteins. The method requires multiple sample preparation steps including the release of glycans from glycoproteins and subsequent derivatization procedures such as permethylation. MS is performed with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). MS of glycans is unable to detect CDG-I, since there are no alterations in the glycoform. MS of intact transferrin , which is modified by the attachment of two complex-type sialylated glycans with minimal microheterogeneity, can be rapidly analyzed, since only immunopurification is needed before loading onto on-line liquid chromatography (LC) MS. ESI of intact transferrin generates ions with 25–45 charges, which requires a wide mass range up to m/z 3000. In addition, parameters must be carefully set for both the measurement and spectral deconvolution in order to identify low-intensity diagnostic signals that might be buried in the chemical noise or lost below the cut-off threshold. Such problems and pitfalls associated with the analysis of intact transferrin have been discussed in detail in a previous report. On the other hand, MALDI-MS of intact transferrin is effective for identifying CDG-I, but the resolving power is not sufficient to detect the altered glycoform profiles that are characteristic of CDG-II.MS can use either intact transferrin, glycans or glycopeptides as the analyte for detecting CDG, but this approach is not widely employed for CDG screening. Barroso et al. recently reported a capillary LC-ESI-MS method to identify CDG and to discriminate between various CDG-II types based on the relative abundance of signature ions. We report herein that the MALDI-MS of tryptic peptides derived from transferrin is a viable alternative to LC-ESI-MS, and the peptide masses can be useful for diagnosing CDG-I and -II serving as reliable biomarkers of CDG.MS of glycopeptides is a standard proteomic method. The tryptic digestion of transferrin followed by MS has been used to characterize the immature glycoforms of CDG-II,Anonymized serum samples were delivered to OWCH to screen patients for suspected CDG. The genetic diagnosis was made before or after identifying the molecular abnormality by MS. Briefly, an affinity column was prepared using a rabbit polyclonal antibody against human transferrin  and a ligand-coupling Sepharose column , and the antibody-coupled Sepharose was recovered from the column. Ten μL of serum or plasma were mixed with a 20-μL slurry of Sepharose in 0.5 mL of phosphate-buffered saline and the resulting solution incubated at 4°C for 30 min. After washing the Sepharose, the transferrin was eluted in 0.1 M glycine–HCl buffer at pH 2.5. The purified transferrin was dissolved in 0.5 mL of 6 M guanidium hydrochloride, 0.25 M Tris–HCl, pH 8.5 and reduced by treatment with 5 mg of dithiothreitol at 60°C for 30 min. A 10 mg portion of iodoacetamide was then added to achieve carbamidomethylation, and the resulting solution was incubated in the dark at room temperature for 30 min. The reagents were removed by a NAP-5 gel filtration column  equilibrated with 0.05 N HCl, and the recovered protein solution was adjusted to pH 8.5 with Tris. Digestion was performed using a mixture of trypsin  and Acromobacter lysylendopeptidase  at 37°C for 12 h. Neither enrichment nor the purification of glycopeptides was carried out. The digest was desalted using a Millipore ZipTip C18 pipette tip and analyzed with a MALDI time-of-flight (TOF) mass spectrometer equipped with a 337-nm wavelength nitrogen laser . The sample matrix was 20 mg/mL of 2,5-dihydroxybenzoic acid dissolved in 50% acetonitrile in water. Measurements were performed for positive ions, and both linear and reflectron TOF modes were used.MS of glycopeptides for glycoform profiling was performed according to a previously reported method, with minor modifications.N-glycosylation sites at Asn413 and Asn611 (Transferrin is abundant (approximately 2 mg/mL) in serum. It contains two d Asn611 , which ad Asn611 , thus alN-acetylneuraminic acids) do not actually reflect the true molar ratio, and the peak for the mono-sialylated species is enhanced as compared with that of the di-sialylated form. This phenomenon is often more evident for the glycopeptide of site-1 than that of site-2. A small signal at m/z 4868.9 corresponds to a fucosylated glycoform at site-2, which is barely detectable at site-1.A MALDI linear TOF mass spectrum of transferrin from a healthy individual is shown in N-glycosylation sites. MALDI reflectron TOF mass spectra from a CDG-I patient (ALG1-CDG) and a healthy individual are shown in m/z 1476.8 and m/z 2515.1 indicate the absence of glycosylation at site-1 and site-2, respectively . Although the precise mechanism underlying the effects of mutations of this gene on glycosylation remains unclear, alterations of the localization of glycosyltransferases in the Golgi apparatus and changes in their enzymatic activity have been suggested to be involved. As shown in m/z 3392, m/z 4140 and m/z 4432) was more evident than decreased galactosylation (m/z 3230 and m/z 4269).ATP6V0A2-CDG is caused by mutations in the gene encoding the a2 subunit of the vacuolar HTRAPPC11, a subunit of the TRAPPIII complex, resulted in a delay of vesicular transport in the Golgi apparatus. The MALDI mass spectrum of TRAPPC11-CDG showed a significant decrease in the extent of sialylation  as well as low-intensity signals indicating decreased galactosylation (m/z 3230 and m/z 4269) (Mutations in /z 4269) , which w In this study, the ratio of the intensities for site-2 glycopeptide ions at m/z 4432 and m/z 4723 was 0.16±0.02  in unaffected individuals. The ratio was significantly elevated for B4GALT1-CDG (0.45), ATP6V0A2-CDG (0.67) and TRAPPC11-CDG (0.46), the only exception being SLC35A2-CDG (0.16), in these mass spectra. It should be noted that the ratio depends on the instrumental parameters involved in the analysis.Regarding sialylation, the level of sialylation to be judged “abnormal” are statistically defined, because the mono-sialo and even asialo species are present in healthy individuals and are enhanced in the MALDI mass spectrum.N-acetylglucosamine caused by a deficiency in GlcNAc transferase II (MGATII-CDG) has also been reported. An exception is MAN1B1-CDG caused by an α-mannosidase I deficiency, which removes α-1,2 mannose units in the Golgi apparatus and is necessary for the formation of complex-type glycans. The hybrid-type glycoforms observed at m/z 3189 and m/z 3351 might be specific biomarkers for MAN1B1-CDG (As shown by the cases described above, most CDG-II types have a decrease in sialylation and/or galactosylation in common as a molecular feature, although a lack of N1B1-CDG . Interes and SLC35C1-CDG, which have an apparently normal transferrin profile. Fucosylation levels in the latter case are effectively evaluated by the MS of immunoglobulin-G, and the MS-based method to detect MOGS-CDG will be described elsewhere. Nevertheless, determining the signature ions of tryptic peptides of transferrin, as shown in Finally, it should be kept in mind that these molecular abnormalities may diminish depending on non-genetic factors such as age, nutrition and treatment. Furthermore, the qualitative or quantitative approach described herein does not allow identification of some types of CDG such as MOGS-CDGN-glycan structure is complicated, the number of key ions are limited, thus facilitating the detection of CDG. Each laboratory should determine its own normal range for these biomarkers especially for the sialylation levels.MALDI-MS of tryptic digests of transferrin offers an easy and reliable method for first-line screening for CDG. Although the


Aggressive fluid administration is recommended in the resuscitation of septic patients. However, the delivery of a rapid fluid bolus might cause harm by inducing degradation of the endothelial glycocalyx. This research aimed to examine the effects of the limited infusion rate of fluid on glycocalyx shedding as measured by syndecan-1 in patients with sepsis-induced hypoperfusion.A prospective, randomized, controlled, open-label trial was conducted between November 2018 and February 2020 in an urban academic emergency department. Patients with sepsis-induced hypoperfusion, defined as hypotension or hyperlactatemia, were randomized to receive either the standard rate (30 ml/kg/h) or limited rate (10 ml/kg/h) of fluid for the first 30 ml/kg fluid resuscitation. Subsequently, the fluid rate was adjusted according to the physician’s discretion but not more than that of the designated fluid rate for the total of 6 h. The primary outcome was differences in change of syndecan-1 levels at 6 h compared to baseline between standard and limited rate groups. Secondary outcomes included adverse events, organ failure, and 90-day mortality.p < 0.001). Patients in the limited-rate group were less likely to received vasopressors  and mechanical ventilation  during the first 6 h. There were no significantly different changes in syndecan-1 levels at 6 h between the two groups . There were no significant differences in adverse events, organ failure outcomes, or mortality between the two groups.We included 96 patients in the intention-to-treat analysis, with 48 assigned to the standard-rate strategy and 48 to the limited-rate strategy. The median fluid volume in 6 h in the limited-rate group was 39 ml/kg (interquartile range [IQR] 35–52 ml/kg) vs. 53 ml/kg (IQR 46–64 ml/kg) in the standard-rate group (In sepsis resuscitation, the limited rate of fluid resuscitation compared to the standard rate did not significantly reduce changes in syndecan-1 at 6 h.http://www.clinicaltrials.in.th/index.php?tp=regtrials&menu=trialsearch&smenu=fulltext&task=search&task2=view1&id=4064Thai Clinical Trials Registry number: TCTR20181010001. Registered 8 October 2018, The online version contains supplementary material available at 10.1186/s40560-020-00515-7. Early and aggressive fluid resuscitation is a mainstay treatment of sepsis-induced hypoperfusion in the emergency department (ED). Fluid resuscitation is typically administered in a bolus to promptly restore mean arterial pressure (MAP) and reverse the microcirculatory derangement. According to the Surviving Sepsis Campaign (SSC) Bundle: 2018 update, 30 ml/kg of crystalloids should be initiated in the first hour for the resuscitation of sepsis-induced hypoperfusion  × 100%). Specified adverse events were monitored during the 6-h intervention period, which included cardiogenic pulmonary edema, new arrhythmia, and the incidence of norepinephrine extravasation. We also recorded protocol adherence and reasons for protocol termination. Serious adverse events were reported to the ethics committee.The primary outcome was differences in change of syndecan-1 levels at 6 h compared to baseline between standard-rate and limited-rate groups. Secondary outcomes were proportions of patients with MAP ≥ 65 mmHg at 1 h and 6 h, 6-h lactate clearance, PaOBlood samples were collected at enrollment and 6 h later to determine plasma syndecan-1 levels. Samples were collected into ethylenediaminetetraacetic acid (EDTA) tubes and stored in a refrigerator before centrifugation followed by storage at – 80 °C. Syndecan-1 levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits . NT-proBNP levels were measured using electrochemiluminescence immunoassay analysis According to earlier research, the standard deviation (SD) of syndecan-1 in septic patients is 109 ng/ml . Therefot test, chi-square test, Fisher’s exact test, or Wilcoxon rank sum test, depending on the types of data. We did not impute missing data. However, the numbers of observations in the analysis are reported. Secondary analysis for the primary outcome included an adjusted analysis for baseline hemodynamics and the concurrent use of vasopressors. We also tested for interactions between the intervention and prespecified subgroups  II score). All analyses were performed using STATA version 16 . Statistical significance was defined as p < 0.05.All analyses were performed according to the intention-to-treat principle. Continuous data are reported as the means with SD or medians with interquartile ranges (IQR), depending on the distribution after normality assessment by visual inspection. Due to highly skewed data, syndecan-1 levels were log-transformed to generate normal distributions and are reported as geometric means with 95% confidence intervals. Categorical data are reported as proportions. A Wilcoxon signed-rank test was used to analyze the change in syndecan-1 from baseline to 6 h. The primary outcome (the differences in change of syndecan-1 level at 6 h compared with that of the baseline between the two groups) was analyzed using linear regression and is reported as a geometric mean ratio (GMR). Secondary outcomes were analyzed with independent From November 2018 to February 2020, 249 patients were screened for eligibility, 146 patients met exclusion criteria, and 5 patients refused to participate in the trial. Ninety-eight patients were randomized to either the standard-rate or the limited-rate group. One patient in each group was excluded from the analysis because they met the exclusion criteria of undergoing emergency surgery. Regarding the primary outcome, syndecan-1 results were missing in two cases of the standard-rate groups and in four cases of the limited infusion rate groups due to administrative reasons and loss to follow-up. In summary, 46 and 44 participants were analyzed for the primary outcome in the standard and limited infusion rate groups, respectively. Forty-eight patients per group were analyzed regarding all other analyses not related to the syndecan-1 test. The patient flow diagram is shown in Fig. p < 0.001). Patients in the limited-rate group were less likely to received vasopressors  compared to the standard-rate group. There was no difference in vasopressor dose between the groups. The use of mechanical ventilation was less frequent in the limited-rate group than in the standard-rate group (23% vs. 42%: p = 0.049). The use of corticosteroids was comparable in both groups (8% vs. 10% p = 0.73), and there was no difference in the use of albumin or time to antibiotics  and limited-rate (n = 44) groups were 265 ng/ml (95% CI 182–388 ng/ml) and 301 (95% CI 206–442 ng/ml) at baseline and 293 ng/ml (95% CI 209–410 ng/ml) and 273 (95% CI 183–408 ng/ml) at 6 h, respectively. There was no significant difference in changes of syndecan-1 level at 6 h  . According to the per-protocol analysis (42 patients in the standard-rate and 38 patients in the limited-rate group), there was no difference between the groups .The geometric means of syndecan-1 in the standard-rate .The median syndecan-1 levels in the standard-rate group were 205 ng/ml (IQR 136–378 ng/ml) and 220 ng/ml (IQR 157–519 ng/ml) at baseline and 6 h, respectively. The median syndecan-1 levels in the limited-rate group were 221 ng/ml (IQR 127–759 ng/ml) and 198 ng/ml (IQR 106–487 ng/ml) at baseline and 6 h, respectively. During the 6-h intervention period, there was no significant different change of syndecan-1 level within the standard-rate and limited rate groups (Protocol adherence was high in both groups since termination of the protocol occurred in only six patients 12%) in the limited-rate group and in five patients (10%) in the standard-rate group. The reasons for protocol termination were the physicians’ decision to change the type of intravenous fluid, the patients exhibited signs of fluid overload, and physicians’ decision to increase the rate of intravenous fluid . The data are summarized in Table Regarding physiological parameters, there were no significantly different changes in 6-h lactate clearance, the proportion of patients with MAP ≥ 65 mmHg at 1 h and 6 h, or the P/F ratio at 6 h. Fluid input per body weight at 24 h was lower in the limited-rate group, but at 72 h, the volume of fluid used was comparable in both groups. There was no difference between the groups with respect to fluid balance at 24 or 72 h, organ failure-free days or hospital length of stay. The 90-day mortality was 18.8% and 31.3% in the limited-rate group and standard-rate group, respectively   Fig. .Fig. 3PIn this randomized controlled trial examining resuscitating patients with sepsis-induced hypoperfusion in the ED, the limited rate of fluid resuscitation compared to the standard rate did not significantly reduce changes in syndecan-1 at 6 h. However, reduced 6-h and 24-h fluid input volumes were observed in the limited-rate group compared to the standard-rate strategy. There was no significant difference in organ failure outcomes, adverse events, or mortality rate between the two groups.Previous studies have reported an association between hypervolemia from rapid fluid administration and glycocalyx shedding as measured by syndecan-1 , 14, 20.Interestingly, in the standard-rate group, the use of vasopressors was more frequent than in the limited-rate group. This could partially be explained by imbalanced baseline characteristics of the participants: there were more patients with hemodynamic instability in the standard-rate group. Moreover, the proportion of mechanically ventilated patients at enrollment in the standard-rate group was higher than that in the limited-rate group , and there was a signal towards mitigating kidney injury in restrictive fluid approach . In the To our knowledge, this study is the first randomized trial comparing different fluid rate strategies during the very early phase of resuscitation in septic patients in the emergency department. The type of resuscitation fluid was controlled provided that different fluid types were associated with different magnitude of glycocalyx damage . HoweverIn patients with sepsis-induced hypoperfusion, the administration of resuscitative fluid with a limited fluid infusion rate did not significantly reduce changes in syndecan-1 at 6 h, but it reduced the volume used during the early resuscitation compared to patients resuscitated using the standard-rate approach.Additional file 1: Table S1. Hemodynamic data of the patients during the intervention. Figure S1. Mean hourly intravenous fluid volume per body weight (ml/kg) during the 6-hour intervention period. The error bars represent the standard deviation. Figure S2. Cumulative number of patients with the need of vasopressors during the 6-hour intervention period. Figure S3. Cumulative number of mechanically-ventilated patients during the 6-hour intervention period.


Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation. P. salmonis is the etiological agent of piscirickettsiosis, an aggressive disease that causes important economic losses in the salmon farming industry [P. salmonis was initially isolated in 1989 from a moribund coho salmon (Oncorhynchus kisutch) during an epizootic event that took place in the south of Chile and was named as the P. salmonis LF-89 strain [P. salmonis has caused serious losses in all farmed salmonid fish species including Atlantic salmon , rainbow trout (Oncorhynchus mykiss), Chinook salmon (Oncorhynchus tshawytscha), pink salmon (Oncorhynchus gorbuscha) and masu salmon (Oncorhynchus masou) [P. salmonis covers a wide geographic range and outbreaks of piscirickettsiosis have been reported among farmed salmonid in Canada, Norway and Ireland; however, mortalities have not been as high as those recorded in Chile [industry ,2,3. Thiindustry . P. salm9 strain ,6,7. Sins masou) . P. salmin Chile . For exain Chile ,13, wherP. salmonis have shown variable levels of virulence that have been linked to the differences in their genetic backgrounds [P. salmonis was first detected by sequencing the ribosomal genes of six P. salmonis isolates [P. salmonis strains that have been often named after the reference strains, LF-89 or EM-90 [P. salmonis strains can be split into two separate genogroups named here as LF-89-like and EM-90-like. The differences that have been reported between these genogroups include geographic distributions, antibiotic susceptibilities and host specificities [P. salmonis genogroups. Field isolates of kgrounds ,15. Geneisolates . The resor EM-90 ,18,19. Wor EM-90 , it is nficities . Moreoveficities  showed tP. salmonis in infected fish tissues has depended on phenotypic and molecular methods such as the culture of P. salmonis on a nutritive media followed by the Gram and Giemsa stain technique [P. salmonis in infected fish tissues, none of them can guarantee the unique presence of the bacterium in the tissue samples [P. salmonis genogroups, an alternative method is the DNA sequencing of the rRNA gene portions, which is usually performed by PCR amplification followed by Sanger sequencing. While this approach is highly reliable and discriminatory, it is time-consuming and costly, and therefore not suitable for most diagnostic laboratories, especially when several hundred clinical isolates are to be identified. For reference laboratories or large epidemiologic studies, a more rapid and inexpensive approach may be required to discriminate between the P. salmonis genogroups. Historically, the identification of echnique ,24,25, cechnique ,27, realechnique ,29 and iechnique . Even th samples . Further samples ,32. ThusXapI for the amplification and digestion steps, respectively. This PCR-RFLP is a rapid, sensitive and cost-effective method to differentiate between the LF-89-like and EM-90-like genogroups. This assay was initially validated by comparing the restriction patterns of 13 P. salmonis strains and further validated by analyzing 57 unknown field samples. Our results showed the PCR-RFLP assay to be a useful tool to identify the P. salmonis genogroups in the field tissue samples without the need of microbiological cultures and bacterial isolation.In this study, we developed a new PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay based on universal 16S rRNA primers and the restriction enzyme P. salmonis available at public databases (March 2020), we selected 76 complete non-redundant 16S rDNA sequences to develop a PCR-RFLP method for the specific identification of the P. salmonis genogroups. Thirty-eight of the sequences were representatives of the EM-90-like genotype and 38 were representatives of the LF-89-like genotype. To select an appropriate restriction enzyme for the PCR-RFLP assay, we follow the pipeline shown in XapI, SfaNI, Hin4II, MluCI, AciI, FaiI and SetI, which produced predicted banding patterns that enabled us to distinguish between the genotypes , which could be relevant for its commercial availability and mass use. This enzyme generates three informative and easily recognizable bands in the 16S rRNA gene fragment of the EM-90-like strains  and four in the 16S rDNA gene fragment of the LF-89-like strains . Upon gel electrophoresis, the RFLPs obtained for the reference strains LF-89 and EM-90 showed perfect matches with those predicted by the in silico digestion with the XapI enzyme  obtained from the tissues of dead or moribund fish with clinical sinology consistent with piscirickettsiosis. The tissues from the infected fish were collected in seawater farms located in three southern regions of Chile , which was previously reported as specific for the P. salmonis 16S rDNA [XapI enzyme resulted in 31 patterns characteristic of the EM-90-like strains and five patterns characteristic of the LF-89-like strains  . In sample PS18259-5  and Vibrio (19.5%), whereas the Piscirickettsia genus was less abundant (3.8%). In the rest of the samples, the most abundant OTUs belonged to the Piscirickettsia genus, although variable percentages of OTUs belonging to other genera were also detected. The Aeribacillus, Anoxybacillus, Pseudomonas, Vibrio, Corynebacterium and Aliibrivio genera were associated with all the field samples and accounted for 25.8% of the relative abundance in the 10 samples. The field samples resolved as containing mixtures of bacterial DNA showed the characteristic detected . We seles (OTUs)  using a s (OTUs) . Eleven s (OTUs) , with PiS18259-5 , the 16SS18700-4 , the mosP. salmonis. Our PCR-RFLP assay is a rapid and cost-effective method, which allows to clearly differentiate the genogroups in the P. salmonis strains and also in the field samples. P. salmonis genotyping could be challenging when relying on isolates since the slow growth rate of P. salmonis in artificial media requires that isolates be cultured for several days to visualize the colonies. Moreover, culture media used to grow P. salmonis are highly nutritive and non-selective, thus they can easily be contaminated with microorganisms (bacteria and fungi) from the environment or those already present in the fish tissues. In this study, we successfully developed a simple genotyping method to identify the genogroups of P. salmonis genogroups relied on the Multilocus sequence typing (MLST) system based on the genetic variation present in 10 P. salmonis housekeeping loci [P. salmonis isolates into two groups that were genetically distinct from the Canadian isolates, and confirmed the existence of at least two different P. salmonis genogroups. In that work, bacteria were isolated from sub-acute or chronic outbreaks of piscirickettsiosis that took place between the years 2011 and 2012 from two geographical areas, Puerto Montt and the east coast of Chiloe Island [P. salmonis isolates recovered from Salmo salar, Oncorhynchus kisutch and Oncorhynchus mykiss from two Chilean regions between the years 2008 and 2015. Their results supported the existence of two P. salmonis genetic groups, but also described possible divergent clades. While MLST is considered to be a highly reliable and discriminatory typing procedure, it is technically demanding, time-consuming and costly [P. salmonis genogroups, the present PCR-RFLP assay based on universal primers and the restriction enzyme XapI provides a method that can be applied to a large number of samples during annual outbreaks without the need of microbiological cultures.Previous genotyping methods aiming to differentiate between the ing loci ,34. Thise Island . Recentld costly . Hence, P. salmonis by the method described by Corbeil et al. [P. salmonis in the samples using a previously reported PCR-RFLP assay [P. salmonis was present in those samples since the PmaCI digestion still revealed the characteristic pattern of this bacterium and the XapI digestion generated the characteristic EM-90-like pattern , it is tempting to speculate that the EM-90-like P. salmonis has become more prevalent than the LF-89-like genogroup. However, further analyses need to be performed, such as genotyping samples along an entire year, sampling tissues from different host species and sampling a wider geographical area, to bring light to this question.Regarding the correlation between the a et al.  reporteda et al.  noted thP. salmonis cultures were inoculated by picking a fresh colony from an Austral-SRS agar plate into 5 mL of an Austral-SRS medium. P. salmonis cultures were grown at 18 °C with shaking at 180 rpm [600) of 0.05 in 5 mL of an Austral-SRS medium and incubated at 18 °C. The absorbance was measured periodically in an Infinite® 200 PRO NanoQuant (Tecan®) until the mid-exponential growth phase was reached. The strains used in this study are LF-89 (ATCC VR-1361), EM-90  and nine field isolates , which are named here as PSCGR01, CGR02, Ps8079A, Ps2192A, Ps8942B, Ps11091B, Ps12201A, Ps18627A and Ps19647A. Further, strains NVI5692, NVI5892 and NVI5896 (kindly donated by Dr. Hanne Winther-Larsen) were also part of this work. LF-89, EM-90, PSCGR01, CGR02, Ps8079A, Ps8942B and Ps11091B were previously sequenced by our group, and their genome sequences are available at the NCBI Genome database under the accession numbers SAMN02469424, SAMN10437718, SAMN04309069, SAMN04309076, SAMN13898195, SAMN13898197 and SAMN13898198, respectively. The genogroup of the other four strains was characterized in this study. The strains were stored in cryopreservation vials at −80 °C and were revived by streaking a loopful of the frozen culture onto an Austral-SRS agar plate ,24 and i 180 rpm . After 4P. salmonis were retrieved from the NCBI_Genome database [P. salmonis from Greengenes [P. salmonis genogroups, we selected only those sequences that contain the 16S rRNA gene universal primers 27F and 1492R [P. salmonis 16S rDNA sequences that were previously selected. To select those restriction enzymes that generate easily differentiable restriction patterns between the LF-89-like and EM-90-like genogroups of P. salmonis, we designed a perl script that compares the bands of the digestion patterns produced by each of the 152 enzymes between the genogroups. If the digestion of the 16S rDNA sequences of a strain belonging to the EM-90-like genogroup produced a banding pattern that matched exactly that of an LF-89-like strain, and there were no additional bands that enabled to differentiate between the digestion patterns, then the enzyme was discarded  and the DNA integrity was verified using agarose gel electrophoresis. The DNA was stored at −20 °C until used. Bacterial DNA was purified from 1 mL of mid-exponential phase growth cultures using the DNeasy Blood and Tissue Kit for DNA (Qiagen). The bacterial culture samples were centrifuged for 10 min at 8000× www.sernapesca.cl), which consists of periodic diagnostic tests to identify the presence of P. salmonis in fish from farm centers. The tissues were extracted from fresh mortalities of the day (less than 12 h). Using a scalpel, a cut was made in the lateral zone, and then five mm3 pieces of brain, liver and/or muscle were dissected from the fish. The organs were placed in a tube with 70% ethanol in a 1:10 ratio (sample/ethanol). The samples were kept at 4 ± 0.5 °C or lower temperatures. The total DNA was extracted from the fish tissue samples using the E.Z.N.A Tissue DNA Kit . Briefly, 30 mg of each tissue was homogenized and transferred to a microcentrifuge tube containing 200 μL of TL buffer, OB protease solution was added and the samples were incubated at 55 °C in a shaking water bath for 3 h. Then, the samples were centrifuged at 12,000× g, and the supernatant was transferred to a new tube. After the BL buffer was added, the mix was incubated at 70 °C for 10 min and finally 220 µL of 100% ethanol was added. The samples were transferred to the HiBind® DNA Mini Column and centrifuged at maximum speed for 1 min at 12,000× g. After the sequential addition of the HBC and DNA wash buffer followed by centrifugation, the DNA was obtained by elution with 200 μL of elution buffer previously heated to 70 °C. The concentration and purity were measured using a NanoQuant Spectrophotometer . The fish samples were collected according to the requirements of the Specific Sanitary Program for Surveillance and Control of Piscirickettsiosis implemented by the Chilean Fisheries and Aquaculture Center , 10 µM of each primer and 8.5 μL of nuclease-free water. The following PCR conditions were used: initial denaturation at 95 °C for 10 min, 30 cycles of 95 °C for 60 s, 60 °C for 30 s and 72 °C for 90 s, with a final extension at 72 °C for 10 min. The amplification products were visualized on 1% agarose gels by ethidium bromide staining and UV illumination. The PCR products from the 16S rRNA gene amplification of the P. salmonis isolates were purified and sequenced in Macrogen . The sequences of the 16S rRNA genes were deposited in GenBank and the accession numbers are given in The 16S rRNA gene was amplified from 50 ng of genomic DNA using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) . The reahttp://tree.bio.ed.ac.uk/software/figtree).The quality of the 16S rRNA gene sequences was corroborated using the BioEdit v7.0.5.3 software . The seqP. salmonis strains and 57 fish tissues (field samples) as described in P. salmonis in the field samples were verified as described in Mandakovic et al. [PmaCI  for 15 min at 37 ℃ followed by agarose gel electrophoresis of the restriction fragments on a 2% gel stained with ethidium bromide. The fragments were visualized in a UV transilluminator and photographed. Then, the PCR amplification products from the P. salmonis strains and field samples were subjected to a XapI enzymatic digestion. The reactions were carried out in 15 μL volumes, which contained 5 μL of the PCR product, 1.5 μL of enzyme buffer, 1 U of each enzyme and 8 μL of nuclease-free water, for 30 min at 37 °C. The restriction fragments were visualized by electrophoresis in 2% agarose gels stained with 0.5 µg/mL of ethidium bromide for 30 min at room temperature. The 16S rRNA gene was amplified from the genomic DNA samples of 13 c et al. . BrieflyThe total DNA (fish + pathogen DNA) extracted from the field samples was amplified using a bacteria-specific primer set with 27F (5′-GAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCGCGGCKGCTG-3′), flanking the hypervariable regions V1–V3 of the 16S rRNA gene  with a bThe 16S rDNA raw amplicon sequences were processed and analyzed following the previously described protocols ,62. Brie


Pseudomonas aeruginosa (P. aeruginosa) is the major pathogenic bacterium colonizing CF lungs at the end of the lifetime of CF patients. This in vitro study uses a P. aeruginosa biofilm model under partly cystic fibrosis conditions, with a sampling of volatile extracellular metabolites. The gas sampling was done with thin-film microextraction (TFME) and commercial polydimethylsiloxane (PDMS) films, whereas the analysis of loaded films was done by gas chromatography coupled to quadrupole mass spectrometry and thermodesorption (TD-GC-qMS). For this purpose, two commercially available films were characterized by means of thermogravimetry coupled to a qMS with atmospheric pressure photo ionization (TG-APPI-qMS), regarding homogeneity and temperature stability. The selected film was cleaned using a method developed in this study. The TD-GC-qMS method was successfully used for standards of volatile metabolites which were known to be produced by P. aeruginosa. Limits of detection and quantification of the method for middle and less polar compounds in low nanomolar range (0.5 nM and 1.5 nM) were achieved. The developed method was finally applied to investigate the extracellular volatile metabolites produced by biofilms of the strain P. aeruginosa DSM 50071 under aerobic and anaerobic conditions. In sum, eleven metabolites could be found under both conditions. Furthermore, it was shown in this study that different oxygen conditions (aerobic and anaerobic) resulted in emitting different extracellular volatile metabolites. Specific metabolites, like 1-undecene (aerobic) and 2-undecanone (anaerobic), could be identified. The results are promising, in that the biofilm model may be applicable for the identification of P. aeruginosa under clinical conditions. Furthermore, the model could be the basis for studying extracellular volatile metabolites from different mono- or co-cultures of various bacteria, as well as the implementation of pulmonary conditions, like these in CF lungs. This possibility allows the development of a non-invasive “at-bedside” breath analysis method for CF patients in focus of various bacterial infections.Cystic fibrosis (CF) is an autosomal recessive inherited disease which leads to a production of thickened mucus in the airways. These conditions are conducive to poly-microbial infections, like chronic lung infection, in which The online version of this article (10.1007/s00216-020-02529-4) contains supplementary material, which is available to authorized users. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that represents a common causative agent of chronic lung infection resulting in a progressive decline of pulmonary function in CF patients + as a function of time  and 2-methylbutanal (2)) can be detected with TD-GC-qMS. Furthermore, it is observed that Gaussian peaks can be obtained, except for dimethyl sulphide. Furthermore, 2-nonanone (7) can separate from 1-undecence (8). This has already been described as a critical separation problem by Zscheppank et al. [6) can also be baseline-separated from 2-nonanone (7). All chosen metabolites are successfully detected as Gaussian peaks with the developed analytical method, with exception of dimethyl sulphide.Figure k et al. . In addiσ method of Kaiser and Specker [For a more accurate characterization of the TD-GC-qMS method, the method detection and quantification limits were determined. The standards listed in Table  Specker . With thP. aeruginosa. To test the general use of our method, the LODs were determined without matrix. Therefore, these values are of course only approximate, since neither the nutrient medium nor the biofilm was considered as matrix. But even if we would spike the biofilm with stable isotopic compounds of these analytes, these LODs would be only approximations, because the conditions in the breath air are completely different.The substances listed in Table The in vitro biofilm model was checked with the multi-standard used for method development. For this purpose, 100 μL of the multi-standard with a concentration of 500 μM for each model substance was inoculated onto the nutrient medium of the biofilm model. The test was carried out for both aerobic and anaerobic conditions with LB-Lennox and potassium nitrate–supplemented LB-Lennox medium, respectively. The chromatograms obtained, including the assignment of the model substances by means of the NIST database, are shown in Fig. P. aeruginosa DSM 50071 were investigated. Both conditions (aerobic and anaerobic) were examined as before. The TICs of these two analyses are shown in Fig. After analysis of the multi-standard in the agar medium, the volatile metabolites of the bacterial strain 11), 2-methyl-quinoxaline (12), 1-undecene (8), decyloxirane (13), methylpyrazine (14), cyclododecane (15), propanoic acid (16) and butanoic acid (17), were detected which were observed only under aerobic conditions. Three metabolites, namely 2-undecanone (18), 2-nonanone (7) and 1-methoxy-2-propanone (19), were identified that could only be observed under anaerobic conditions. The retention time (SD less than 5 s) and the NIST score (greater than 80%) were used as criteria for identification. Table P. aeruginosa in previous publications. These are the metabolites acetic acid, 1-undecene, methylpyrazine, propanoic acid, 2-undecanone and 2-nonanone [P. aeruginosa cultures, 2-methyl-quinoxalines, decyloxirane, cyclododecane, butanoic acid and 1-methoxy-2-propanone.Figure nonanone –33. HoweIn addition to the identification, a differentiation based on the identified metabolites was possible. To compare the two models regarding differences in metabolic products, the total ion chromatograms were closely investigated in the retention time range from 17.5 to 21.5 min. Figure 8) is formed only under aerobic conditions, whereas 2-undecanone (18) is formed only under anaerobic conditions. To assess the repeatability of the method, including biological variability, nine biological replicates were studied on two different days. The retention time, the area and the NIST database score between the biological replicates were compared. 1-undecene elutes at a retention time of 18.21 min ± 0.01 min. Furthermore, the area of 1-undecene has a relative standard deviation of 7.53%. This is a very small deviation in view of the high biological variability. The NIST score averages 94% with a standard deviation of 1%. Analogously, the same parameters for the 2-undecanone were calculated. This results in a retention time of 21.09 min ± 0.01 min and a relative standard deviation of the area of 9.44%. The NIST database score is 96% for 2-undecanone with a deviation of 1%. In addition, the variance in peak heights of the two metabolites was examined. Analysis of the nine biological replicates revealed a difference in heights of 13.2% and 12.4% for the 1-undecene and 2-undecanone, respectively. Considering the biological variability, the system deviations by 13% of the heights is highly acceptable.In Fig. P. aeruginosa was developed. The model could be the basis for studying extracellular volatile metabolites from various mono- and co-cultures under pulmonary conditions, like these in CF lungs. A methodology for sampling of extracellular volatile metabolites, using TFME, was developed and applied to the in vitro biofilm model. Thermal analysis helped to investigate the selected sorbent material by means of quality control. Furthermore, it could be shown that the ionization of cyclic siloxanes with APPI is possible in a TG-qMS system. The developed in vitro model was successfully validated using standards and real bacterial biofilms. The analysis of model metabolites was used to determine the LOD and LOQ in low nanomolar ranges. Eleven metabolites, for strain P. aeruginosa DSM 50071, were found with the developed methodology and it could be shown that differentiation between aerobic and anaerobic growing conditions based on volatile metabolites is possible.In this study, an in vitro model to analyse the extracellular volatile metabolites formed under biofilm conditions of P. aeruginosa, like strains PAO1 and FRD1, as well as fresh clinical isolates. The focus is on the identification of specific metabolites of mucoid strains, which mainly grow in CF lungs. Furthermore, the in vitro model should be improved in perspective of CF lung infection using artificial sputum medium, real sputum medium and multiple bacteria suspensions, to imitate the lung of CF patient in different stages of the disease. With this method, maybe volatile metabolites of P. aeruginosa and hopefully other bacteria could be determined as biomarkers. These biomarkers may then be detected by a non-invasive “at-bedside” breath target analysis method to detect severe lung infections with P. aeruginosa of CF patients at an early stage.Prospectively, the developed methodology, including in vitro model, sampling and analytical system, should be used for a comparison of well-characterized clinical isolates from ESM 1(PDF 788 kb)